Performing Department
(N/A)
Non Technical Summary
Challenges in the production of hydrolysate and other milk-derived products center on the efficient generation of a high-yield product. Enzymes enable reaction processes to take place under less harsh conditions than chemical-only methods, increase product specificity, and reduce the difficulty of reaction processing and waste cleanup. The use of enzymes is not without its own challenges. The primary use limitations come from the cost of the enzyme, and that enzymes in their purified form are limited to a single batch reaction. Recovery of enzyme from the reaction mixture for potential reuse in dairy processing is difficult since the substrates, products, and the enzymes all possess similar physical properties. Other cost-drivers are tied to the need to inactivate the enzyme to stop a reaction, and removal of the enzyme from the final product if the enzyme can cause an allergic or toxic response.To enable the reuse of enzymes, studies have investigated ultrafiltration membranes scaled to retain the larger-size enzymes and simultaneously function as the first purification step, orbinding enzymes to the membranes to combine the reaction and separation processes. These membrane-based solutions demonstrated efficiency gains but suffered from increased enzyme deactivation through shear and thermal stress, enzyme leakage into the product, and membrane fouling. As an alternative, enzymes were immobilized on solid supports such as glass, agarose, and silica. Immobilizing enzymes to these supports enabled improved regulation of the reactions, increased enzyme stability, reduced pressure drop issues in the reactors, improved separation of product from enzyme, and reduced contamination. However, the value gained by these improvements was offset by the high price of the enzyme support material.The technology demonstrated in Phase I and to be advanced in the proposed Phase II SBIR utilizes ImmobiZyme, Guild BioSciences patented immobilization platform where active components, such as enzymes, are chemically bound to inactive biodegradable support materialsconsisting primarily oflow-cost or recycled bio-waste. These raw materials offer significant cost savings from traditional immobilization supports, enable higher-quality enzyme preparations, and simplify post-reaction recycling/reuse processes, while retaining the advantages of more expensive support materials.The overall aim of the Phase II project is to develop ImmobiZyme into a market-ready product for whey and milk hydrolysis. Phase I efforts demonstrated that these pellets retained more than 90% of enzyme activity through 350 hours of continuous reaction with a per batch material loss of less than 5%. Producing cost-effective ImmobiZyme pellets at-scale is essential to the success of the project. IE must remain active and whole through multiple consecutive batch reactions, have a simple and efficient recovery process, not carry microbial contamination to subsequent reactions, and meet regulatory requirements for contact with materials for human consumption. Our plan is to start optimization based on performance requirements recommended through our relationships with 4 U.S. producers. Transitioning this proof of concept material to a product will be achieved by: 1) improving the durability of the pellets so the per batch material loss is less than 1%, 2) defining temperature-related performance limitations and optimums, 3) development of an effective pellet cleaning process that maximizes pellet reuse while minimizing batch-to-batch contamination, 4) developing a cost-efficient, uniform, quality, pilot-scale production process capable of kilogram scale batches, 5) demonstrate the performance of the new material internally and with the support of external facilities, and 6) proceeding towards regulatory approval for the ImmobiZyme platform. Results from Phase II will support the technical and economic advantages of using ImmobiZyme pellets as a drop-in replacement for current whey or milk hydrolysis enzymes.
Animal Health Component
0%
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Goals / Objectives
Goal 1: Demonstrate modifications to the Phase I formulation to produce an immobilized enzymepellet with improved durability, broad temperature range, reduced material costs, and equivalent or better performance than the Phase I materials.Objective 1: Phase II ImmobiZyme pellets retain/recover >99% pellet mass across 30 batches.Objective 2: Define Phase II pellet performance at reaction temperatures from 4 °C-80 °C.Goal 2: Develop a pellet cleaning process to reduce inter-batch contamination.Objective 3: Efficacy of individual process steps to remove bacteria from pellets of immobilized enzymewhile maximizing retained activity will be determined.Objective 4: Verify a cleaning process that produces a 3-log reduction in viable bacteria on pellets, while retaining >95% pellet enzyme activity pre-to-post wash.Goal 3: Assess regulatory requirements for the immobilized enzyme platform, ImmobiZymeObjective 5: Identify gaps in existing data required for GRAS or other regulatory approval.Objective 6: Select and perform additional testing or evaluations as necessary to verify the non-toxic nature of the ImmobiZyme platform.Goal 4: Develop pilot scale production line capable of producing 1 kg to 10 kg per batch.Objective 7: Pellet enzyme activity, size uniformity, and physical stability will be within 5% of lab-scale design parameters.Goal 5: Evaluate pilot-scale materials.Objective 8: Pellet meets previously defined performance requirements through at least 30 batches.Objective 9: Analyze and compare results from outside lab evaluations. Understand where potential deviations happened and prepare for any final refinements.
Project Methods
The overall aim of the Phase II project is to develop a market-ready product for whey and milk hydrolysis. The immobilized enzymes must remain active and whole through multiple consecutive batch reactions, have a simple and efficient recovery process, not carry microbial contamination to subsequent reactions, and meet regulatory requirements for contact with materials for human consumption. The project will focus on improving Phase I pellet performance: improving the durability of the pellets, developing, and validating a pellet cleaning process to prevent microbial cross-contamination, and defining the temperature range limits for reactions. This will be followed by manufacturing pellets at a pilot-scale, which includes defining quality control processes and determining methods to reduce production costs. This will operatein parallel with an investigation into the unmet requirements for regulatory approval of the ImmobiZyme platform and perform tests as recommended for regulatory approval. Finally, pilot-scale reactions will be performed at our facility will be comapared to independent performance evaluations at external labs. Success of this project will be measured through the achievement of the milestones described below.Milestone 1: ImmobiZyme pellets retain/recover >99% pellet mass across 30 consecutive test reaction batches.The durability of the pellet formulation will be optimized through changes in the mass ratios of the primary components of the pellet, and changes to the application of crosslinker in terms of concentration, reaction time, and temperature. The initial physical stability of the pellets will be measured via agitation in an 8000D mixer/mill (Spex SamplePrep). After shaking, the beads will be separated using a 0.5 mm sieve and the retained beads weighed. TEM imaging will be used to determine additional quantitative correlations between formulation, pore structure, and durability. The most durable candidate pellets determined by this testing will be further evaluated using a repeated batch study using both buffer solutions and reaction solutions. Pellets will undergo 30 consecutive reaction batches of 6-24 hours each and be evaluated for pellet mass. Successful pellets will retain more than 99% of their mass on a batch-to-batch basis through the entire process.Milestone 2: Define Phase II pellet performance at reaction temperatures from 4-80°C.Enzyme substrate solutions will be equilibrated at reaction temperatures between 4-60°C, prior to the addition of enzyme. The reaction temperatures will be held through the reaction of the assay and samples will be returned to room temperature by submersion in a water bath prior to the separation/deactivation of the enzyme. Following this testing, the relationship between the activity of the pellets and time at a temperature will be evaluated by immersing the pellets into PBS solutions at temperatures between 30-80°C for up to 100 hours. The pellets will be removed from the heated solutions and evaluated for their activity vs. an unexposed material.Milestone 3: Efficacy of individual process steps to remove bacteria from ImmobiZyme pellets while maximizing retained activity will be determined.Immobilized enzyme pellets in substrate solutions will be contaminated with representative microbial contaminants common to dairy processing facilities and incubated for up to 24 hours. The pellets will be separated and washed using common Clean in Place (CIP) chemicals and conditions. Following the wash step the pellets will be transferred to fresh test solution and incubated to allow any retained bacteria to grow. At the end of the second incubation period, pellets will be collected and placed in an ultrasonic bath to dislodge any retained bacteria. Samples of the test solution and the buffers solution from the bath will be serially diluted onto agar plates to quantify the number of cells that survived cleaning steps. The various cleaning steps will be ranked based on the log reduction in viable bacteria.Milestone 4: Verify a cleaning process that produces a 3-log reduction in viable bacteria on pellets, while retaining >95% pellet enzyme activity pre-to-post wash.As data is generated, different wash steps will be studied in a serial fashion to optimize the cleaning of the pellets with respect to the overall retained activity. Based on these results a cleaning process will be developed and verified to meet the bacterial reduction and retained enzyme activity requirements.Milestone 5: Identify gaps in existing data required for GRAS or other regulatory filings.GBS will be working with Lee Enterprises Consulting (LEC) to evaluate the regulatory pathway and assess ImmobiZyme and its manufacturing process to identify and fill gaps in existing data needed to meet regulatory requirements keeping in mind the changing regulatory landscape.Milestone 6: Select and perform additional testing or evaluations as necessary to verify the non-toxic nature of the ImmobiZyme platform.We anticipate that some toxicology testing will be required to support a future regulatory filing. Based on the recommendations from LEC we will seek out accredited vendors to perform various toxicology tests to maximize the information we will get from toxicology testing.Milestone 7: Develop pilot scale production line capable of producing 1 kg to 10 kg per batch.Producing ImmobiZyme pellets at the 1-10 kg scale per batch will require adapting the methods and formulations defined in the Phase I effort and refined earlier in this Phase II effort to appropriately scaled equipment and the development of a dedicated manufacturing process. The pilot-scale process will be incrementally integrated and monitored to define essential points of control along the production pathway. Identifying these critical points such as the rate of pellet addition and the optimum time/temperature for enzyme addition will identify necessary areas and processes for QC monitoring.Milestone 8: Verify pilot-scale production materials meet lab-scale performance requirements: with pellet enzyme activity, size uniformity, and physical stability within 5% of lab-scale design parameters and demonstrate resilience through 30 test batches.Immobilized enzyme pellets produced at the pilot-scale will be evaluated based on their enzyme activity, size uniformity, and physical stability and compared to the performance of materials produced at the lab-bench scale. Batch-to-batch evaluation of the pilot-scale materials will take place in 1-10 L reactors. The process parameters will follow optimum reaction conditions identified previously and utilize the pellet cleaning procedures defined earlier in this work. ImmobiZyme pellet performance will be evaluated in terms of total substrate conversion, retained pellet activity, and recovered pellet mass over 30 consecutive batch runs of 6-24 hours depending on reaction temperatures and the substrate. Pellets sampled between batches will be evaluated for enzyme activity to confirm that any losses are within previously established limits. The reaction substrate conversion will be analyzed and compared to commercial enzyme counterparts.Milestone 9: Analyze and compare results from outside lab evaluations. Determine where potential deviations happened and their causes to make final refinements to production.Following the success of the in-house pilot-scale product evaluations we will reach out to potential hydrolysate manufacturers, and after securing appropriate material transfer agreements, send them kg quantities of the pilot scale material for their evaluation. The test conditions to be used will be selected by these companies to match their standards for new material validation testing. This effort will not only provide outside lab verification of our material performance, but also provide insight into other use conditions and potentially serve to increase interest in our product.